PROJECT SUMMARY The aim of this grant is to elucidate the thiol redox modification of the Cu importer, CTR1 as a vital link between Cu transporters, reactive oxygen species (ROS)-dependent VEGFR2 signaling and reparative angiogenesis. ROS derived from NADPH oxidase (NOX) act as signaling molecules to promote VEGF-induced angiogenesis in endothelial cells (ECs) and reparative neovascularization. The fundamental question remains, how diffusible ROS can activate specific redox signaling to enhance therapeutic angiogenesis? The signaling function of ROS acts through oxidation of reactive Cys residues in proteins to generate ?Cysteine sulfenic acid (Cys-OH)? (sulfenylation), which is involved in disulfide bond formation and redox signaling. Copper (Cu), an essential micronutrient, also plays an important role in angiogenesis via unknown mechanisms. The major Cu entry pathway is via the Cu importer, CTR1, which has only one cytosolic Cys189 in the highly conserved C- terminal triad, HCH190. The co-investigator of this grant reported that the HCH190 triad acts as a loose ?plug? for Cu entry, and is essential for Cu-induced CTR1 internalization (regulatory endocytosis) which protects against excess Cu-induced toxicity in HEK cells. However, the mechanistic linkage between CTR1 and VEGF-induced ROS signaling in mediating angiogenesis in ECs and its in vivo role are entirely unknown. Based on our preliminary data, we hypothesize that VEGF induces sulfenylation of CTR1 at Cys189 via NOX-derived ROS, which drives: 1) CTR1 binding to VEGFR2 and their subsequent co-internalization required for activating sustained VEGFR2 signaling in a Cu transport-independent manner; and 2) Cu entry-dependent activation of Cu target proteins in ECs. This in turn promotes full angiogenesis and neovascularization in ischemic diseases. Aim 1 will characterize the VEGF-induced Cys oxidation of CTR1 and determine its role in angiogenic responses in human and mouse ECs. Aim 2 will determine the mechanisms by which Cys-oxidized CTR1 activates VEGFR2 signaling and Cu entry-dependent activation of Cu targets in ECs. Aim 3 will determine the in vivo significance of endothelial CTR1 function in ROS-dependent reparative neovascularization and address underlying mechanisms using mice hindlimb ischemia and wound healing models. We will use various innovative reagents, including biotin-labelled Cys-OH trapping probe; BiFC-based protein-protein interaction in situ and live cell imaging, cell surface biotinylation; and gene transfer of EC-targeted various CTR1 mutants; newly-developed inducible EC-specific CTR1-/- mice and CRISPR/Cas9-generated CTR1 Cys oxidation- defective knock-in mutant mice. Highly innovative ICP-Mass Spec, X-ray fluorescence microscopy, Cu fluorescence probe will be used to analyze intracellular Cu in cells and tissues. Our proposal will provide novel insights into Cys oxidized CTR1 as a potential therapeutic target for ischemic cardiovascular diseases.